Characterization and molecular docking study of cathepsin L inhibitory peptides (SnuCalCpIs) from Calotropis procera R. Br.

Propeptides, released from the autocatalytic activation of its zymogen, are potential inhibitors against proteases involved in cancer cell invasion and migration. Our research team previously obtained novel propeptides (SnuCalCpIs) from transcriptome analysis of the medicinal plant Calotropis procera R. Br. and reported them as promising candidates for cancer therapeutics due to their cathepsin L inhibition activity. In the present study, inhibitory activity among SnuCalCpIs was compared with inhibition efficiency and verified by in silico molecular docking analysis. Only SnuCalCpI03 and SnuCalCpI15, expressed in Escherichia coli, showed inhibitory activity against cathepsin L as competitive inhibitors, and the half-maximal inhibitory concentrations (IC50) values of 2.1 nM and 1.6 nM, respectively. They were stable below 70 °C, maintaining more than 90% inhibitory activity over a wide range of pH (2.0-10.0), except at the isoelectric point (pI). The template-based docking simulation models showed that SnuCalCpI02, SnuCalCpI12, and SnuCalCpI16 could not interact with the substrate-binding cleft of cathepsin L even though they possessed the same conserved domain. In contrast, SnuCalCpI03 and SnuCalCpI15 interacted with cathepsin L along the propeptide binding loop and substrate-binding cleft, resulting in obstruction of substrate access to the active site.

Origin of the natural variation in the storage of dietary carotenoids in freshwater amphipod crustaceans.

Carotenoids are diverse lipophilic natural pigments which are stored in variable amounts by animals. Given the multiple biological functions of carotenoids, such variation may have strong implications in evolutionary biology. Crustaceans such as Gammarus amphipods store large amounts of these pigments and inter-population variation occurs. While differences in parasite selective pressure have been proposed to explain this variation, the contribution of other factors such as genetic differences in the gammarid ability to assimilate and/or store pigments, and the environmental availability of carotenoids cannot be dismissed. This study investigates the relative contributions of the gammarid genotype and of the environmental availability of carotenoids in the natural variability in carotenoid storage. It further explores the link of this natural variability in carotenoid storage with major crustacean immune parameters. We addressed these aspects using the cryptic diversity in the amphipod crustacean Gammarus fossarum and a diet supplementation protocol in the laboratory. Our results suggest that natural variation in G. fossarum storage of dietary carotenoids results from both the availability of the pigments in the environment and the genetically-based ability of the gammarids to assimilate and/or store them, which is associated to levels of stimulation of cellular immune defences. While our results may support the hypothesis that carotenoids storage in this crustacean may evolve in response to parasitic pressure, a better understanding of the specific roles of this large pigment storage in the crustacean physiology is needed.

Hyperbaric oxygen therapy accelerates wound healing in diabetic mice by decreasing active matrix metalloproteinase-9.

Diabetic foot ulcers are characterized by hypoxia. For many patients, hyperbaric oxygen (HBO) therapy is the last recourse for saving the limb from amputation, for which the molecular basis is not understood. We previously identified the active form of matrix metalloproteinase-9 (MMP-9) as responsible for diabetic foot ulcer's recalcitrance to healing. Transcription of mmp-9 to the inactive zymogen is upregulated during hypoxia. Activation of the zymogen is promoted by proteases and reactive oxygen species (ROS). We hypothesized that the dynamics of these two events might lead to a lowering of active MMP-9 levels in the wounded tissue. We employed the full-thickness excisional db/db mouse model to study wound healing, and treated the mice to 3.0 atm of molecular oxygen for 90 minutes, 5 days per week for 10 days in an HBO research chamber. Treatment with HBO accelerated diabetic wound healing compared to untreated mice, with more completed and extended reepithelialization. We imaged the wounds for ROS in vivo with a luminol-based probe and found that HBO treatment actually decreases ROS levels. The levels of superoxide dismutase, catalase, and glutathione peroxidase-enzymes that turn over ROS-increased after HBO treatment, hence the observation of decreased ROS. Since ROS levels are lowered, we explored the effect that this would have on activation of MMP-9. Quantitative analysis with an affinity resin that binds and pulls down the active MMPs exclusively, coupled with proteomics, revealed that HBO treatment indeed reduces the active MMP-9 levels. This work for the first time demonstrates that diminution of active MMP-9 is a contributing factor and a mechanism for enhancement of diabetic wound healing by HBO therapy.

Procaspase-3 Overexpression in Cancer: A Paradoxical Observation with Therapeutic Potential.

Many anticancer strategies rely on the promotion of apoptosis in cancer cells as a means to shrink tumors. Crucial for apoptotic function are executioner caspases, most notably caspase-3, that proteolyze a variety of proteins, inducing cell death. Paradoxically, overexpression of procaspase-3 (PC-3), the low-activity zymogen precursor to caspase-3, has been reported in a variety of cancer types. Until recently, this counterintuitive overexpression of a pro-apoptotic protein in cancer has been puzzling. Recent studies suggest subapoptotic caspase-3 activity may promote oncogenic transformation, a possible explanation for the enigmatic overexpression of PC-3. Herein, the overexpression of PC-3 in cancer and its mechanistic basis is reviewed; collectively, the data suggest the potential for exploitation of PC-3 overexpression with PC-3 activators as a targeted anticancer strategy.

PI3K/Akt Cooperates with Oncogenic Notch by Inducing Nitric Oxide-Dependent Inflammation.

The PI3K/Akt signaling pathway, Notch, and other oncogenes cooperate in the induction of aggressive cancers. Elucidating how the PI3K/Akt pathway facilitates tumorigenesis by other oncogenes may offer opportunities to develop drugs with fewer side effects than those currently available. Here, using an unbiased in vivo chemical genetic screen in Drosophila, we identified compounds that inhibit the activity of proinflammatory enzymes nitric oxide synthase (NOS) and lipoxygenase (LOX) as selective suppressors of Notch-PI3K/Akt cooperative oncogenesis. Tumor silencing of NOS and LOX signaling mirrored the antitumor effect of the hit compounds, demonstrating their participation in Notch-PI3K/Akt-induced tumorigenesis. Oncogenic PI3K/Akt signaling triggered inflammation and immunosuppression via aberrant NOS expression. Accordingly, activated Notch tumorigenesis was fueled by hampering the immune response or by NOS overexpression to mimic a protumorigenic environment. Our lead compound, the LOX inhibitor BW B70C, also selectively killed human leukemic cells by dampening the NOTCH1-PI3K/AKT-eNOS axis.

Highly Conserved Arg Residue of ERFNIN Motif of Pro-Domain is Important for pH-Induced Zymogen Activation Process in Cysteine Cathepsins K and L.

Pro-domain of a cysteine cathepsin contains a highly conserved Ex2Rx2Fx2Nx3Ix3N (ERFNIN) motif. The zymogen structure of cathepsins revealed that the Arg(R) residue of the motif is a central residue of a salt-bridge/H-bond network, stabilizing the scaffold of the pro-domain. Importance of the arginine is also demonstrated in studies where a single mutation (Arg → Trp) in human lysosomal cathepsin K (hCTSK) is linked to a bone-related genetic disorder "Pycnodysostosis". In the present study, we have characterized in vitro Arg → Trp mutant of hCTSK and the same mutant of hCTSL. The R → W mutant of hCTSK revealed that this mutation leads to an unstable zymogen that is spontaneously activated and auto-proteolytically degraded rapidly. In contrast, the same mutant of hCTSL is sufficiently stable and has proteolytic activity almost like its wild-type counterpart; however it shows an altered zymogen activation condition in terms of pH, temperature and time. Far and near UV circular dichroism and intrinsic tryptophan fluorescence experiments have revealed that the mutation has minimal effect on structure of the protease hCTSL. Molecular modeling studies shows that the mutated Trp31 in hCTSL forms an aromatic cluster with Tyr23 and Trp30 leading to a local stabilization of pro-domain and supplements the loss of salt-bridge interaction mediated by Arg31 in wild-type. In hCTSK-R31W mutant, due to presence of a non-aromatic Ser30 residue such interaction is not possible and may be responsible for local instability. These differences may cause detrimental effects of R31W mutation on the regulation of hCTSK auto-activation process compared to altered activation process in hCTSL.

Inhibition of pro-/active MMP-2 by green tea catechins and prediction of their interaction by molecular docking studies.

Matrix metalloproteinases (MMPs) play a crucial role in developing different types of lung diseases, e.g., pulmonary arterial hypertension (PAH). Green tea polyphenolic catechins such as EGCG and ECG have been shown to ameliorate various types of diseases including PAH. Our present study revealed that among the four green tea catechins (EGCG, ECG, EC, and EGC), EGCG and ECG inhibit pro-/active MMP-2 activities in pulmonary artery smooth muscle cell (PASMC) culture supernatant. Based on the above, we investigated the interactions of pro-/active MMP-2 with the green tea catechins by computational methods. In silico analysis revealed a strong interaction of pro-/active MMP-2 with EGCG/ECG, and galloyl group has been observed to be responsible for this interaction. The in silico analysis corroborated our experimental observation that EGCG and ECG are active in preventing both the proMMP-2 and MMP-2 activities. Importantly, these two catechins appeared to be better inhibitors for proMMP-2 in comparison to MMP-2 as revealed by gelatin zymogram and also by molecular docking studies. In many type of cells, activation of proMMP-2 occurs via an increase in the level of MT1-MMP (MMP-14). We, therefore, determined the interactions of MT1-MMP with the green tea catechins by molecular docking analysis. The study revealed a strong interaction of MT1-MMP with EGCG/ECG, and galloyl group has been observed to be responsible for the interaction.

Anti-wrinkle and anti-whitening effects of jucá (Libidibia ferrea Mart.) extracts.

Skin aging is a natural process of the human body that may be accelerated due to extrinsic causes. Libidibia ferrea, popularly known as jucá, is a small tree, which possesses an abundant phenolic composition with potential antioxidant and enzymatic inhibition activities. Thus, this work aimed to investigate the anti-wrinkle and anti-whitening potentials of jucá trunk bark (LFB) and pod (LFP) extracts. A comprehensive analysis of LFB and LFP phenolic composition was accomplished by means of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Effects on skin degradation were assessed by inhibitory enzymatic activity against elastase, hyaluronidase and collagenase through colorimetric assays. Cellular viability in B16F10 and primary fibroblasts were determined by Trypan Blue exclusion assay. Anti-melanogenic effects on B16F10 cells were evaluated using cellular tyrosinase, melanin content, western blot, and RT-qPCR analyses. Inhibition of matrix metalloproteinase-2 and metalloproteinase-9 (MMP-2 and MMP-9) was determined by gelatin zymography and western blot methodologies. LC-MS/MS analyses of LFB and LFP extracts allowed the characterization of 18 compounds, among them, flavonoids, phenolic acids, and secoridoids. Additionally the pod and trunk bark compositions were compared. Hyaluronidase inhibitory activity for both extracts, LFB (IC50 = 8.5 ± 0.8 µg/mL) and LFP (IC50 = 16 ± 0.5 µg/mL), was stronger than standard rutin (IC50 = 27.6 ± 0.06). Pro-MMP-2 was significantly inhibited by both extracts. LFB and LFP decreased the melanin content in B16F10 due to tyrosinase inhibitory activity. L. ferrea extracts has high potential as a cosmetic ingredient due to its anti-wrinkle and depigmentant effects.

Proteome-wide covalent ligand discovery in native biological systems.

Small molecules are powerful tools for investigating protein function and can serve as leads for new therapeutics. Most human proteins, however, lack small-molecule ligands, and entire protein classes are considered 'undruggable'. Fragment-based ligand discovery can identify small-molecule probes for proteins that have proven difficult to target using high-throughput screening of complex compound libraries. Although reversibly binding ligands are commonly pursued, covalent fragments provide an alternative route to small-molecule probes, including those that can access regions of proteins that are difficult to target through binding affinity alone. Here we report a quantitative analysis of cysteine-reactive small-molecule fragments screened against thousands of proteins in human proteomes and cells. Covalent ligands were identified for >700 cysteines found in both druggable proteins and proteins deficient in chemical probes, including transcription factors, adaptor/scaffolding proteins, and uncharacterized proteins. Among the atypical ligand-protein interactions discovered were compounds that react preferentially with pro- (inactive) caspases. We used these ligands to distinguish extrinsic apoptosis pathways in human cell lines versus primary human T cells, showing that the former is largely mediated by caspase-8 while the latter depends on both caspase-8 and -10. Fragment-based covalent ligand discovery provides a greatly expanded portrait of the ligandable proteome and furnishes compounds that can illuminate protein functions in native biological systems.

Expression of surface-associated 82kDa-proMMP-9 in primary acute leukemia blast cells inversely correlates with patients' risk.

With its ability to degrade extracellular matrix proteins and activate growth factors and cytokines, matrix metalloproteinase (MMP)-9 is an important regulator of cell function. Previously, we reported that myeloid leukemic cells express a unique 82kDa-proMMP-9 variant on their cell surface that is not affected by its natural inhibitor. In this study, we generated monoclonal antibodies that specifically recognize 82kDa-proMMP-9. Flow cytometry analysis using these antibodies revealed significant surface expression of 82kDa-proMMP-9 in monocytes, but minimal amounts in T and B cells isolated from peripheral blood of nine healthy donors and 22 patients with acute myeloid leukemia (AML). In all AML patients, blasts expressed 82kDa-proMMP-9 at levels of 4%-46%, with significantly higher levels in patients with a better risk defined according to National Comprehensive Cancer Network (NCCN) guidelines (ρ = -0.748, p < 0.001) and favorable phenotype according to the French-American-British classification (p = 0.02) compared with patients with adverse prognoses. Receiver operating characteristic curve analysis confirmed the diagnostic accuracy of 82kDa-proMMP-9 measurement in AML blasts (area under the curve: 0.893 [0.739-1.000], p = 0.019). It led us to define a cutoff value of 11.5% for identifying patients with lower NCCN risk (p = 0.005) and with a tendency toward a higher probability of response to anthracycline-based therapy (p = 0.109) and increased event-free survival (p = 0.24). Thus, 82kDa-proMMP-9 expression on blasts may represent a novel independent marker of prognosis in patients with AML.

A single CRD C-type lectin from Eriocheir sinensis (EsLecB) with microbial-binding, antibacterial prophenoloxidase activation and hem-encapsulation activities.

C-type lectins (CTLs) exist widely in crustaceans. To date, thirteen CTLs have been reported in crustaceans, and play significant roles in pathogen recognition, encapsulation of hemocytes and antimicrobial activity in the innate immune response. Based on the initial expressed sequence tags (EST) of a hepatopancreatic cDNA library, a novel CTL, designated as EsLecB, with a 470 bp open reading frame encodes a polypeptide of 156 amino acids, including a signal peptide of 19 amino acid residues and one carbohydrate-recognition domain of 131 aa residues, was cloned from the crustacean Eriocheir sinensis. By qRT-PCR analysis, EsLecB was detected in all tested tissues, and showed highest expression in hemocytes, hepatopancreas and heart. The expression of EsLecB was up-regulated following injections of PAMPs or bacteria. The recombinant protein (rEsLecB) expressed in Escherichia coli had a calcium-independent but carbohydrate-dependent microbial-binding and microbial-agglutinating, microorganism growth inhibitory and hem-encapsulation activities. Moreover, the rEsLecB could stimulate the activation of prophenoloxidase in vitro. These results indicated that EsLecB, as an antibacterial pattern recognition receptor is involved in innate immunity, and may act as an upstream detector of the prophenoloxidase activating system, which can detect pathogen invasion in E. sinensis.

Lipopolysaccharide and ß-1,3-glucan-binding protein (LGBP) bind to seaweed polysaccharides and activate the prophenoloxidase system in white shrimp Litopenaeus vannamei.

Lipopolysaccharide and ß-1,3-glucan-binding protein (LGBP), important pattern recognition proteins (PRPs), recognize lipopolysaccharide (LPS) and ß-1,3-glucan (ßG), known as pathogen-associated molecular patterns (PAMPs), and subsequently trigger innate immunity. Several seaweed polysaccharides and seaweed extracts increase immune parameters and resistance to pathogens. Here, we constructed the expression vector pET28b-LvLGBP and transferred it into Escherichia coli BL21 (DE3) for protein expression and to produce the recombinant protein LGBP (rLvLGBP) in white shrimp Litopenaeus vannamei. We examined the binding of rLvLGBP with seaweed-derived polysaccharides including alginate, carrageenan, fucoidan, laminarin, Gracilaria tenuistipitata extract (GTE), and Sargassum duplicatum extract (SDE), and examined the phenoloxidase activity of shrimp haemocytes incubated with a mixture of rLvLGBP and each polysaccharide. We also examined the binding of rLvLGBP with LPS and ßG, and the phenoloxidase activity of shrimp haemocytes incubated with a mixture of rLvLGBP and LPS (rLvLGBP-LPS) or a mixture of rLvLGBP and ßG (rLvLGBP-ßG). An ELISA binding assay indicated that rLvLGBP binds to LPS, ßG, alginate, carrageenan, fucoidan, laminarin, GTE, and SDE with dissociation constants of 0.1138-0.1770 µM. Furthermore, our results also indicated that the phenoloxidase activity of shrimp haemocytes incubated with a mixture of rLvLGBP and LPS, ßG, alginate, carrageenan, fucoidan, laminarin, GTE, and SDE significantly increased by 328%, 172%, 200%, 213%, 197%, 194%, 191%, and 197%, respectively compared to controls (cacodylate buffer). We conclude that LvLGBP functions as a PRP, recognizes and binds to LPS, ßG, alginate, carrageenan, fucoidan, laminarin, GTE, and SDE, and subsequently leads to activating innate immunity in shrimp.

Characterization of the effects of C-terminal pro-sequence on self-inactivation of Stereum purpureum endopolygalacturonase I.

Endpolygalacturonase I from Stereum purpureum has been identified as a causative substance for the silver-leaf disease in apples. It possesses a unique pro-sequence in the C-terminal region that lacks endpolygalacturonases from any other origin. In this study, we analyzed and compared enzymatic characteristics between pro-form (pro-endoPG I) and mature form processed by V8 protease (endoPG I) and described the suppression activity of the pro-sequence. Of note, the optimal pH for pro-endoPG I activity shifted to pH 4.0 from pH 4.5-5.0 of endoPG I. The kinetic parameters indicated that the activity inhibition resulted from a pH-independent decrease of substrate affinity and pH-dependent deterioration of velocity by the pro-sequence. Analysis of site-directed mutations within pro-endoPG I showed that its α-helical structure includes two glutamates (E364 and E366) and alanine (A365), and its orientation by prolines (especially P348) in the pro-sequence played a significant role in its suppression activity. As for mutations in the mature domain, a marked reduction of suppression was observed for enzymes with mutations in H150, R220 and K253, indicating that the pro-sequence interacts with the active cleft by a few ionic bonds.

Food-mediated modulation of immunity in a phytophagous insect: An effect of nutrition rather than parasitic contamination.

Inherent to the cost of immunity, the immune system itself can exhibit tradeoffs between its arms. Phytophagous insects face a wide range of microbial and eukaryotic parasites, each activating different immune pathways that could compromise the activity of the others. Feeding larvae are primarily exposed to microbes, which growth is controlled by antibiotic secondary metabolites produced by the host plant. The resulting variation in abundance of microbes on plants is expected to differentially stimulate the insect antimicrobial immune defenses. Under the above tradeoff hypothesis, stimulation of the insect antimicrobial defenses is expected to compromise immune activity against eukaryote parasites. In the European grape berry moth, Eupoecilia ambiguella, immune effectors directed towards microbes are negatively correlated to those directed towards eukaryotic parasites among host plants. Here, we hypothesize this relationship is caused by a variable control of the microbial community among host plants by their antibiotic metabolites. To test this hypothesis, we first quantified antimicrobial activity in berries of several grape varieties. We then measured immune defenses of E. ambiguella larvae raised on artificial diets in which we mimicked levels of antimicrobial activity of grape berries using tetracycline to control the abundance of growing microbes. Another group of larvae was raised on artificial diets made of berry extracts only to control for the effect of nutrition. We found that controlling microbe abundance with tetracycline in diets did not explain variation in the immune function whereas the presence of berry extracts did. This suggests that variation in immune defenses of E. ambiguella among grape varieties is caused by nutritional difference among host plants rather than microbe abundance. Further study of the effects of berry compounds on larval immune parameters will be needed to explain the observed tradeoff among immune system components.

Zingiber cassumunar ROXb. and its active constituent inhibit MMP-9 direct activation by house dust mite allergens and MMP-9 expression in PMA-stimulated human airway epithelial cells.

BACKGROUND: House dust mite (HDM) induced matrix metalloproteinase (MMP)-9 plays a role in asthma. Zingiber cassumunar Roxb. (Phlai in Thai) has been used in folk medicine for asthma treatment. OBJECTIVE: We investigated effects of Phlai and its constituent (E)-4-(3',4'-dimethoxyphenyl)but-3-en-1-ol (compound D) on the cleavage of pro- MMP-9 by HDM. The effects of these compounds on phorbol 12-myristate 13-acetate (PMA)- induced MMP-9 gene and protein expression in airway epithelial cells (NCI-H292) were also investigated. METHODS: Pro-MMP-9 was directly activated in vitro with HDM in the presence or absence of the ethanolic extracts of Phlai or compound D for 1 hour. The amount of activated MMP-9 was determined using gelatin zymography. To study the cellular response of Phlai, NCI-H292 cells were pretreated with crude Phlai extracts or compound D for 2 hours, and then the cells were stimulated with PMA for 48 hours. The mRNA RT-PCR and Western blotting, respectively. MMP-9 activity was determined by gelatin zymography. RESULTS: Crude Phlai extracts (0.25 - 2.0 mg/ml) and compound D (0.5 - 4.0 mg/ml) inhibited pro- MMP-9 cleavage by HDM. Furthermore, crude Phlai extracts (100 mg/ml) and compound D, at concentrations of 50 and 100 mg/ml, attenuated the PMA-induced MMP-9 gene and expression in NCI-H292 cells. These compound also suppressed MMP-9 release from PMA-induced NCI-H292 cells. CONCLUSION: The crude ethanolic extract of Z. cassumunar and its active constituent compound D inhibited the cleavage of pro-MMP-9 by HDM. They also inhibited PMA-induced MMP-9 gene and protein synthesis in human airway epithelial cells.

The roles of Na⁺/K⁺-ATPase α-subunit gene from the ridgetail white prawn Exopalaemon carinicauda in response to salinity stresses.

Na(+)/K(+)-ATPase (NAK) is one important transporter protein and plays a key role in maintaining osmotic homeostasis in low and high salinity acclimation in variety of crustacean species. The ridgetail white prawn Exopalaemon carinicauda is an euryhaline and economic shrimp species in China, but it remains unclear about its mechanism of salinity adaption. In this study, a full-length of Na(+)/K(+)-ATPase α-subunit (α-NAK) cDNA was cloned from E. carinicauda by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of α-NAK was of 3680 bp, containing an open reading frame (ORF) of 3030 bp encoding a polypeptide of 1009 amino acids with the predicted molecular weight of 112.27 kDa. Eight transmembrane domains and two sites of phosphorylation and ATP binding were identified in E. carinicauda α-NAK. BLAST analysis revealed that the sequence of α-NAK amino acids of E. carinicauda shared more than 75% homologies with those of other crustacean. Real time quantitative RT-PCR analysis indicated that E. carinicauda α-NAK gene could be detected in all the tested tissues with highest expression level in gill. The expression profiles of E. carinicauda α-NAK transcripts were analyzed in gill and hepatopancreas tissues after salinity stresses. The results showed that the expression level of E. carinicauda α-NAK gene in both gill and hepatopancreas reached peak at different time after low and high salinity stresses, and showed different expression profiles. The expression profiles of proPO transcripts in gills after salinity stresses also indicated α-NAK and proPO played synergistic actions for salinity responses in E. carinicauda. These results indicated that E. carinicauda α-NAK involved in stress responses against salinity.

Fingerprinting of gelatinase subtypes for different topographic regions on non-retaining placenta of Holstein cows.

The contribution of matrix metalloproteinases (MMP) to timely discharge of the placenta from bovine uterus at parturition is yet inconclusive, partly because of the presence of multiple MMP forms in situ. In the current study, the expression of different gelatinase subtypes on non-retaining placentas of Holstein cows was fingerprinted by using gelatin zymography. Different topographic regions on the placenta were measured separately, including the placentome-like structure and the fetal and maternal sides of interplacentomal placenta, all sampled from the central and peripheral areas of the placenta, respectively. The spontaneously ruptured umbilical cords were cross-sectioned as fetus end, middle and placenta end also for separate measurement. Body fluids including blood samples from the parturient cows, their neonatal calves and umbilical cord, as well as fetal fluids and the first colostrum were measured concomitantly. Results showed multiple forms of gelatinases subtypes in the placenta tissues and body fluids, including neutrophil gelatinase-associated lipocalin (NGAL)-MMP-9 complex, both the latent and active forms of MMP-2 and MMP-9; of them, the latent forms were much more abundantly and frequently expressed than the active forms. NGAL-MMP-9 complex was more prevalently present in the body fluids than in the placenta tissues. No distinguishable pattern of the expression of any gelatinase subtype was observed among the placentome-like structure, interplacentomal placenta and umbilical cord, or between fetal and maternal sides. Nonetheless, for interplacentomal placenta, proMMP-9 expression was higher in the central than in the peripheral area. In addition, proMMP-2 expression was higher in the rupture end (fetus end) than the placenta end of the umbilical cord. In conclusion, the current validated gelatin zymography detected a gradient proMMP-9 expression on the non-retaining placenta of cows in reverse to the proximity to the umbilical insertion point, and a gradient proMMP-2 expression on a section of the umbilical cord in reverse to the proximity to the rupture site, suggesting roles played by gelatinases in normal discharge of the placenta at term.

Whey peptides prevent chronic ultraviolet B radiation-induced skin aging in melanin-possessing male hairless mice.

Whey proteins or peptides exhibit various actions, including an antioxidant action, an anticancer action, and a protective action against childhood asthma and atopic syndrome. The effects of orally administered whey peptides (WPs) on chronic ultraviolet B (UVB) radiation-induced cutaneous changes, including changes in cutaneous thickness, elasticity, wrinkle formation, etc., have not been examined. In this study, we studied the preventive effects of WPs on cutaneous aging induced by chronic UVB irradiation in melanin-possessing male hairless mice (HRM). UVB (36-180 mJ/cm(2)) was irradiated to the dorsal area for 17 wk in HRM, and the measurements of cutaneous thickness and elasticity in UVB irradiated mice were performed every week. WPs (200 and 400 mg/kg, twice daily) were administered orally for 17 wk. WPs inhibited the increase in cutaneous thickness, wrinkle formation, and melanin granules and the reduction in cutaneous elasticity associated with photoaging. Furthermore, it has been reported that UVB irradiation-induced skin aging is closely associated with the increase in expression of matrix metalloproteinase (MMP), vascular endothelial growth factor (VEGF), Ki-67-, and 8-hydroxy-2'-deoxyguanosine (8-OHdG)-positive cells. WPs also prevented increases in the expression of MMP-2 and pro-MMP-9, VEGF, and Ki-67- and 8-OHdG-positive cells induced by chronic UVB irradiation. It was found that WPs prevent type IV collagen degradation, angiogenesis, proliferation, and DNA damage caused by UVB irradiation. Overall, these results demonstrate the considerable benefit of WPs for protection against solar UV-irradiated skin aging as a supplemental nutrient.

Fisetin inhibits matrix metalloproteinases and reduces tumor cell invasiveness and endothelial cell tube formation.

Matrix metalloproteinases (MMPs) play an important role in tissue remodeling during normal physiological situations and pathological implications such as tumor invasion and metastasis. MMP inhibitors were screened from extracts of medicinal herbs by an enzymatic assay using the MMP-14 catalytic domain. Among samples tested, a methanol extract of the root of Dalbergia odorifera T. CHEN (Leguminosae) showed the strongest inhibitory activity. The inhibitory component was purified through fractionation methods and identified as fisetin, abundant in many fruits and vegetables. In addition to inhibition of MMP-14, fisetin inhibits MMP-1, MMP-3, MMP-7, and MMP-9, more efficiently than a naturally occurring MMP inhibitor tetracycline. Fisetin dose-dependently inhibits proliferation of fibrosarcoma HT-1080 cells and human umbilical vascular endothelial cells (HUVECs), MMP-14-mediated activation of proMMP-2 in HT-1080 cells, invasiveness of HT-1080 cells, and in vitro tube formation of HUVECs. Therefore, fisetin could be valuable as a chemopreventive agent against cancer and a lead compound for development of therapeutic MMP inhibitors.

Cloning of prophenoloxidase from hemocytes of the blue crab, Callinectes sapidus and its expression and enzyme activity during the molt cycle.

The arthropods cuticle undergoes dramatic morphological and biochemical changes from being soft to hardness through each molting process. Prophenoloxidase (PPO) known as a key enzyme in the arthropod innate immune system involved in the melanization reaction, has been related with the initial shell-hardening process, specifically in the sclerotization of the protein matrix in the new cuticle. Since hemocytes have been reported as the main PPO source in arthropods, the transport of hemocyte PPO into the newly laid, soft cuticle has been proposed for shell-hardening occurring during and immediately after ecdysis. In order to define the role of hemocyte PPO in the shell-hardening of crustaceans, the full-length cDNA sequence (2806 nt) of hemocytes PPO of the blue crab Callinectes sapidus (CasPPO-hemo) is isolated using degenerate PCR and 5'-3' RACE. CasPPO-hemo encodes a putative PPO (672 aa) showing three hemocyanin domains: N, M, and C in order and two copper binding sites (CuA & CuB). The sequence analysis identifies the putative CasPPO-hemo as zymogen which requires the cleavage at the N-terminus for its activation. Hemocyte extract (CasHLS) contains the PO, the activity of which depends on the in vitro activation of trypsin. The expression levels of CasPPO-hemo are kept constant during the molt cycle. The increase in the number of hemocytes at early premolt correlates with the elevated PO activity, while at late premolt, the increment in hemocyte numbers does not reflect on the PO activity. The functional importance of the changes in the levels of CasHLS-PO activity during molt cycle is discussed in relation to cuticle hardening process.